More than 1/3 of the world?s population is anemic, making the process of erythropoiesis central to human health and disease. The maturation of a committed erythroid progenitor to a functional erythrocyte is characterized by a global decline in transcription and progressive chromatin condensation that ultimately culminates in enucleation. Although these processes occur in parallel, the molecular mechanisms that coordinate them are unknown. We hypothesize that Setd8 is a critical regulator of mammalian erythropoiesis that functions to coordinate the fundamental processes of transcriptional repression and chromatin condensation. Setd8 is the sole enzyme in mammals capable of mono-methylating H4K20 (H4K20me1), and is expressed at significantly higher levels in CD71+ erythroid cells than in any other cell- or tissue-type. In addition, Setd8 has functions that are independent of its methyltransferase activity. The overarching goal of this proposal is to elucidate the role of Setd8 in the regulation of mammalian erythropoiesis. Data from our laboratory demonstrates that conditional erythroid Setd8 deletion results in severe defects in primitive erythropoiesis, with visible anemia at E9.5 and death from anemia by ~E12.5. Erythroblasts from these embryos fail to undergo the typical semi-synchronous maturation observed in erythroblasts from control embryos and have a profound defect in nuclear condensation. The goal of Aim 1 is to delineate the function of Setd8 during mammalian erythropoiesis. In Aim 1, in vivo analyses of conditional Setd8 disruption will be coupled with knockdown of Setd8 in human CD34+ hematopoietic stem and progenitor cells to gain a comprehensive understanding of the function of Setd8 in mammalian erythropoiesis. Preliminary data from our laboratory further demonstrates that Setd8 functions as a transcriptional repressor in erythroid cells, and that the Gata2 locus is a direct Setd8 target. Knockdown of Setd8 leads to increased Gata2 expression, with loss of H4K20me1 and increased H4Acetylation in the Gata2 locus. Although Gata2 is a key regulator of erythroid differentiation, Gata2 overexpression alone does not account for the severity of the Setd8 phenotype, especially the profound impairment in nuclear condensation. The Condensin II complex recognizes H4K20me1 and mediates both transcriptional repression and higher order chromatin condensation. Interestingly, Setd8 and a member of the Condensin II complex co-occupy key regulatory regions of the Gata2 locus. The goal of Aim 2 is to delineate the molecular mechanism(s) underlying the role of Setd8 in erythropoiesis. In Aim 2 we will determine the mechanisms by which Setd8 regulates key erythroid loci, and the degree to which the Setd8-H4K20me1-Condensin II pathway mediates transcriptional repression and chromatin condensation in erythroid cells. Taken together, the studies in this proposal will delineate the function of a novel regulator of erythropoiesis and provide critical insights into pathways involved in both inherited and acquired human disease.